Splicing variant of human membrane-type matrix metalloproteinase-5 (MT-MMP5-L)

ABSTRACT

The MT-MMP5-L polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing MT-MMP5-L polypeptides and polynucleotides in therapy, and diagnostic assays for such.

FIELD OF THE INVENTION

[0001] This invention relates to newly identified polypeptides andpolynucleotides encoding such polypeptides, to their use in therapy andin identifying compounds which may be agonists, antagonists and/orinhibitors which are potentially useful in therapy, and to production ofsuch polypeptides and polynucleotides.

BACKGROUND OF THE INVENTION

[0002] The drug discovery process is currently undergoing a fundamentalrevolution as it embraces ‘functional genomics’, that is, highthroughput genome- or gene-based biology. This approach is rapidlysuperseding earlier approaches based on ‘positional cloning’. Aphenotype, that is a biological function or genetic disease, would beidentified and this would then be tracked back to the responsible gene,based on its genetic map position.

[0003] Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of potential interest from themany molecular biology databases now available. There is a continuingneed to identify and characterize further genes and their relatedpolypeptides/proteins, as targets for drug discovery.

[0004] Membrane-Type Matrix Metalloproteinases (MT-MMPs) are a newfamily of transmembrane matrix metalloproteinases. At present, there arefour human MT-MMPs published in the literature (Sato, II. et al., Nature370:61-65, 1994; Will, H. and Hinzmann, B. Eur. J. Biochem. 231:602-608,1995; Takino, T. et al., J. Biol. Chem. 270:23013-23020; Puente, X. S.et al., Cancer Res. 56:944-949, 1996). Recently, a fifth MT-MMP(MT-MMP-5) has been published for mouse (Pei, D., J. Biol. Chem.274:8925-8932, 1999). Also, a splice variant for human MT-MMP-5 in whichthe catalytic domain is missing has been patented (U.S. Pat. No.5,937,508).

[0005] MT-MMPs function as both a receptor and as an activator forcertain MMPs and serve to localize extracellular matrix protcolysis atthe pericellular region. MT-MMPs have been shown to play a role inmetastasis and have been identified in numerous carcinomas. An MT-MMPhas also been demonstrated to be involved in Alzheimer's Disease whereit has been found in white matter microglia (Yamada, T. et al., ActaNeuropathol. 90: 421-424, 1995). MT-MMPs may also play a role in theinfiltration of inflammatory cells. By Northern Array Grid Analysis,MT-MMP5-L expression was detected in cerebellum and kidney. A multipletissue Northern blot was also probed for MT-MMP5-L where it wasidentified in brain, kidney and pancreas. This indicates that theseMembrane-Type Matrix Metalloproteinase have an established, provenhistory as therapeutic targets. Clearly there is a need foridentification and characterization of further members of Membrane-TypeMatrix Metalloproteinase family which can play a role in preventing,ameliorating or correcting dysfunctions or diseases, including, but notlimited to, 1) kidney-related diseases including diabetes, polycystickidney disease, renal failure, etc., 2) brain-related diseases includingAlzheimer's disease, stroke multiple sclerosis, neurodegenerativediseases, etc., 3) cardiovascular diseases including restenosis,myocardial infarction, dilated cardiomyopathy, atherosclerosis, etc.,and 4) others such as inflammation and cancer.

SUMMARY OF THE INVENTION

[0006] The present invention relates to MT-MMP5-L, in particularMT-MMP5-L polypeptides and MT-MMP5-L polynucleotides, recombinantmaterials and methods for their production. In another aspect, theinvention relates to methods for using such polypeptides andpolynucleotides, including the treatment of 1) kidney-related diseasesincluding diabetes, polycystic kidney disease, renal failure, etc., 2)brain-related diseases including Alzheimer's disease, stroke multiplesclerosis, neurodegenerative diseases, etc., 3) cardiovascular diseasesincluding restenosis, myocardial infarction, dilated cardiomyopathy,atherosclerosis, etc., and 4) others such as inflammation and cancer,hereinafter referred to as “the Diseases”, amongst others. In a furtheraspect, the invention relates to methods for identifying agonists andantagonists/inhibitors using the materials provided by the invention,and treating conditions associated with MT-MMP5-L imbalance with theidentified compounds. In a still further aspect, the invention relatesto diagnostic assays for detecting diseases associated withinappropriate MT-MMP5-L activity or levels.

DESCRIPTION OF THE INVENTION

[0007] In a first aspect, the present invention relates to MT-MMP5-Lpolypeptides. Such peptides include isolated polypeptides comprising anamino acid sequence which has at least 70% identity, preferably at least80% identity, more preferably at least 90% identity, yet more preferablyat least 95% identity, most preferably at least 97-99% identity, to thatof SEQ ID NO:2 over the entire length of SEQ ID NO:2. Such polypeptidesinclude those comprising the amino acid of SEQ ID NO:2.

[0008] Further peptides of the present invention include isolatedpolypeptides in which the amino acid sequence has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, most preferably at least97-99% identity, to the amino acid sequence of SEQ ID NO:2 over theentire length of SEQ ID NO:2. Such polypeptides include the polypeptideof SEQ ID NO:2.

[0009] Further peptides of the present invention include isolatedpolypeptides encoded by a polynucleotide comprising the sequencecontained in SEQ ID NO: 1.

[0010] Polypeptides of the present invention are believed to be membersof the MT-MMP family of polypeptides. They are therefore of interestbecause MT-MMPs have been shown to play a role in metastasis and havebeen identified in numerous carcinomas. An MT-MMP has also beendemonstrated to be involved in Alzheimer's Disease where it has beenfound in white matter microglia (Yamada, T. et al., Acta Neuropathol.90: 421-424, 1995). MT-MMPs may also play a role in the infiltration ofinflammatory cells. These properties are hereinafter referred to as“MT-MMP5-L activity” or “MT-MMP5-L polypeptide activity” or “biologicalactivity of MT-MMP5-L”. Also included amongst these activities areantigenic and immunogenic activities of said MT-MMP5-L polypeptides, inparticular the antigenic and immunogenic activities of the polypeptideof SEQ ID NO:2. Preferably, a polypeptide of the present inventionexhibits at least one biological activity of MT-MMP5-L.

[0011] The polypeptides of the present invention may be in the form ofthe “mature” protein or may be a part of a larger protein such as afusion protein. It is often advantageous to include an additional aminoacid sequence which contains secretory or leader sequences,pro-sequences, sequences which aid in purification such as multiplehistidine residues, or an additional sequence for stability duringrecombinant production.

[0012] The present invention also includes include variants of theaforementioned polypeptides, that is polypeptides that vary from thereferents by conservative amino acid substitutions, whereby a residue issubstituted by another with like characteristics. Typical suchsubstitutions are among Ala, Val, Leu and Ile; among Ser and Thr; amongthe acidic residues Asp and Glu; among Asn and Gln; and among the basicresidues Lys and Arg; or aromatic residues Phe and Tyr. Particularlypreferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 aminoacids are substituted, deleted, or added in any combination.

[0013] Polypeptides of the present invention can be prepared in anysuitable manner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

[0014] In a further aspect, the present invention relates to MT-MMP5-Lpolynucleotides. Such polynucleotides include isolated polyrtucleotidescomprising a nucleotide sequence encoding a polypeptide which has atleast 70% identity, preferably at least 80% identity, more preferably atleast 90% identity, yet more preferably at least 95% identity, to theamino acid sequence of SEQ ID NO:2, over the entire length of SEQ IDNO:2. In this regard, polypeptides which have at least 97% identity arehighly preferred, whilst those with at least 98-99% identity are morehighly preferred, and those with at least 99% identity are most highlypreferred. Such polynucleotides include a polynucleotide comprising thenucleotide sequence contained in SEQ ID NO:1 encoding the polypeptide ofSEQ ID NO:2.

[0015] Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence that has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, to a nucleotidesequence encoding a polypeptide of SEQ ID NO:2, over the entire codingregion. In this regard, polynucleotides which have at least 97% identityare highly preferred, whilst those with at least 98-99% identity aremore highly preferred, and those with at least 99% identity are mosthighly preferred.

[0016] Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence which has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, to SEQ ID NO:1 overthe entire length of SEQ ID NO:1. In this regard, polynucleotides whichhave at least 97% identity are highly preferred, whilst those with atleast 98-99% identify are more highly preferred, and those with at least99% identity are most highly preferred. Such polynucleotides include apolynucleotide comprising the polynucleotide of SEQ ID NO:1 as well asthe polynucleotide of SEQ ID NO:1.

[0017] The invention also provides polynucleotides which arecomplementary to all the above described polynucleotides.

[0018] The nucleotide sequence of SEQ ID NO:1 shows homology with humanMT-MMP3 and mouse MT5-MMP (D. Pei, JBC, 274:8925-8932, 1999). Thenucleotide sequence of SEQ ID NO:1 is a cDNA sequence and comprises apolypeptide encoding sequence (nucleotide 306 to 1901) encoding apolypeptide of 532 amino acids, the polypeptide of SEQ ID NO:2. Thenucleotide sequence encoding the polypeptide of SEQ ID NO:2 may beidentical to the polypeptide encoding sequence contained in SEQ ID NO:1or it may be a sequence other than the one contained in SEQ ID NO:1,which, as a result of the redundancy (degeneracy) of the genetic code,also encodes the polypeptide of SEQ ID NO:2. The polypeptide of SEQ IDNO:2 is structurally related to other proteins of the MT-MMP family,having homology and/or structural similarity with human MT-MMP3 andmouse MT5-MMP (D. Pei, JBC, 274:8925-8932, 1999).

[0019] Preferred polypeptides and polynucleotides of the presentinvention are expected to have, inter alia, similar biologicalfunctions/properties to their homologous polypeptides andpolynucleotides. Furthermore, preferred polypeptides and polynucleotidesof the present invention have at least one MT-MMP5-L activity.

[0020] Polynucleotides of the present invention may be obtained, usingstandard cloning and screening techniques, from a cDNA library derivedfrom mRNA in cells of human brain, using the expressed sequence tag(EST) analysis (Adams, M. D., et al. Science (1991) 252:1651-1656;Adams, M. D. et al., Nature, (1992) 355:632-634; Adams, M. D., et al.,Nature (1995) 377 Supp:3-174). Polynucleotides of the invention can alsobe obtained from natural sources such as genomic DNA libraries or can besynthesized using well known and commercially available techniques.

[0021] When polynucleotides of the present invention are used for therecombinant production of polypeptides of the present invention, thepolynucleotide may include the coding sequence for the maturepolypeptide, by itself; or the coding sequence for the maturepolypeptide in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence, or other fusion peptide portions. For example, amarker sequence which facilitates purification of the fused polypeptidecan be encoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., ProcNatl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotidemay also contain non-coding 5′ and 3′ sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

[0022] Further embodiments of the present invention includepolynucleotides encoding polypeptide variants which comprise the aminoacid sequence of SEQ ID NO:2 and in which several, for instance from 5to 10, 1 to 5, 1 to 3, 1 to 2 or 1, amino acid residues are substituted,deleted or added, in any combination.

[0023] Polynucleotides which are identical or sufficiently identical toa nucleotide sequence contained in SEQ ID NO:1, may be used ashybridization probes for cDNA and genomic DNA or as primers for anucleic acid amplification (PCR) reaction, to isolate full-length cDNAsand genomic clones encoding polypeptides of the present invention and toisolate cDNA and genomic clones of other genes (including genes encodinghomologs and orthologs from species other than human) that have a highsequence similarity to SEQ ID NO:1. Typically these nucleotide sequencesare 70% identical, preferably 80% identical, more preferably 90%identical, most preferably 95% identical to that of the referent. Theprobes or primers will generally comprise at least 15 nucleotides,preferably, at least 30 nucleotides and may have at least 50nucleotides. Particularly preferred probes will have between 30 and 50nucleotides.

[0024] A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than human, may beobtained by a process which comprises the steps of screening anappropriate library under stringent hybridization conditions with alabeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof;and isolating full-length cDNA and genomic clones containing saidpolynucleotide sequence. Such hybridization techniques are well known tothe skilled artisan. Preferred stringent hybridization conditionsinclude overnight incubation at 42° C. in a solution comprising: 50%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH7.6), 5× Denhardt's solution, 10 % dextran sulfate, and 20microgram/ml denatured, sheared salmon sperm DNA; followed by washingthe filters in 0.1×SSC at about 65° C. Thus the present invention alsoincludes polynucleotides obtainable by screening an appropriate libraryunder stringent hybridization conditions with a labeled probe having thesequence of SEQ ID NO:1 or a fragment thereof.

[0025] The skilled artisan will appreciate that, in many cases, anisolated cDNA sequence will be incomplete, in that the region coding forthe polypeptide is cut short at the 5′ end of the cDNA. This is aconsequence of reverse transcriptase, an enzyme with inherently low‘processivity’ (a measure of the ability of the enzyme to remainattached to the template during the polymerisation reaction), failing tocomplete a DNA copy of the mRNA template during 1st strand cDNAsynthesis.

[0026] There are several methods available and well known to thoseskilled in the art to obtain full-length cDNAs, or extend short cDNAs,for example those based on the method of Rapid Amplification of cDNAends (RACE) (see, for example, Frohman et al., PNAS USA 85, 8998-9002,1988). Recent modifications of the technique, exemplified by theMarathon™ technology (Clontech Laboratories Inc.) for example, havesignificantly simplified the search for longer cDNAs. In the Marathon™technology, cDNAs have been prepared from mRNA extracted from a chosentissue and an ‘adaptor’ sequence ligated onto each end. Nucleic acidamplification (PCR) is then carried out to amplify the ‘missing’ 5′ endof the cDNA using a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using‘nested’ primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the known gene sequence). The products of thisreaction can then be analysed by DNA sequencing and a full-length cDNAconstructed either by joining the product directly to the existing cDNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

[0027] Recombinant polypeptides of the present invention may be preparedby processes well known in the art from genetically engineered hostcells comprising expression systems. Accordingly, in a further aspect,the present invention relates to expression systems which comprise apolynucleotide or polynucleotides of the present invention, to hostcells which are genetically engineered with such expression systems andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

[0028] For recombinant production, host cells can be geneticallyengineered to incorporate expression systems or portions thereof forpolynucleotides of the present invention. Introduction ofpolynucleotides into host cells can be effected by methods described inmany standard laboratory manuals, such as Davis et al., Basic Methods inMolecular Biology (1986) and Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989). Preferred such methods include, forinstance, calcium phosphate transfection, DEAE-dextran mediatedtransfection, transvection, microinjection, cationic lipid-mediatedtransfection, electroporation, transduction, scrape loading, ballisticintroduction or infection.

[0029] Representative examples of appropriate hosts include bacterialcells, such as streptococci, staphylococci, E. coli, Streptomyces andBacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 andBowes melanoma cells; and plant cells.

[0030] A great variety of expression systems can be used, for instance,chromosomal, episomal and virus-derived systems, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression systems may containcontrol regions that regulate as well as engender expression. Generally,any system or vector which is able to maintain, propagate or express apolynucleotide to produce a polypeptide in a host may be used. Theappropriate nucleotide sequence may be inserted into an expressionsystem by any of a variety of well-known and routine techniques, suchas, for example, those set forth in Sambrook et al., MOLECULAR CLONING,A LABORATORY MANUAL (supra). Appropriate secretion signals may beincorporated into the desired polypeptide to allow secretion of thetranslated protein into the lumen of the endoplasmic reticulum, theperiplasmic space or the extracellular enviromnent. These signals may beendogenous to the polypeptide or they may be heterologous signals.

[0031] If a polypeptide of the present invention is to be expressed foruse in screening assays, it is generally preferred that the polypeptidebe produced at the surface of the cell. In this event, the cells may beharvested prior to use in the screening assay. If the polypeptide issecreted into the medium, the medium can be recovered in order torecover and purify the polypeptide. If produced intracellularly, thecells must first be lysed before the polypeptide is recovered.

[0032] Polypeptides of the present invention can be recovered andpurified from recombinant cell cultures by well-known methods includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Mostpreferably, high performance liquid chromatography is employed forpurification. Well known techniques for refolding proteins may beemployed to regenerate active conformation when the polypeptide isdenatured during isolation and or purification.

[0033] This invention also relates to the use of polynucleotides of thepresent invention as diagnostic reagents. Detection of a mutated form ofthe gene characterised by the polynucleotide of SEQ ID NO:1 which isassociated with a dysfunction will provide a diagnostic tool that canadd to, or define, a diagnosis of a disease, or susceptibility to adisease, which results from under-expression, over-expression or alteredexpression of the gene. Individuals carrying mutations in the gene maybe detected at the DNA level by a variety of techniques.

[0034] Nucleic acids for diagnosis may be obtained from a subject'scells, such as from blood, urine, saliva, tissue biopsy or autopsymaterial. The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniquesprior to analysis. RNA or cDNA may also be used in similar fashion.Deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to labeled MT-MMP5-Lnucleotide sequences. Perfectly matched sequences can be distinguishedfrom mismatched duplexes by RNase digestion or by differences in meltingtemperatures. DNA sequence differences may also be detected byalterations in electrophoretic mobility of DNA fragments in gels, withor without denaturing agents, or by direct DNA sequencing (e.g., Myerset al., Science (1985) 230:1242). Sequence changes at specific locationsmay also be revealed by nuclease protection assays, such as RNase and S1protection or the chemical cleavage method (see Cotton et al., Proc NatlAcad Sci USA (1985) 85: 4397-4401). In another embodiment, an array ofoligonucleotides probes comprising MT-MMP5-L nucleotide sequence orfragments thereof can be constructed to conduct efficient screening ofe.g., genetic mutations. Array technology methods are well known andhave general applicability and can be used to address a variety ofquestions in molecular genetics including gene expression, geneticlinkage, and genetic variability (see for example: M.Chee et al.,Science, Vol 274, pp 610-613 (1996)).

[0035] The diagnostic assays offer a process for diagnosing ordetermining a susceptibility to the Diseases through detection ofmutation in the MT-MMP5-L gene by the methods described. In addition,such diseases may be diagnosed by methods comprising determining from asample derived from a subject an abnormally decreased or increased levelof polypeptide or mRNA. Decreased or increased expression can bemeasured at the RNA level using any of the methods well known in the artfor the quantitation of polynucleotides, such as, for example, nucleicacid amplification, for instance PCR, RT-PCR, RNase protection, Northernblotting and other hybridization methods. Assay techniques that can beused to determine levels of a protein, such as a polypeptide of thepresent invention, in a sample derived from a host are well-known tothose of skill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

[0036] Thus in another aspect, the present invention relates to adiagonostic kit which comprises:

[0037] (a) a polynucleotide of the present invention, preferably thenucleotide sequence of SEQ ID NO: 1, or a fragment thereof;

[0038] (b) a nucleotide sequence complementary to that of (a);

[0039] (c) a polypeptide of the present invention, preferably thepolypeptide of SEQ ID NO:2 or a fragment thereof; or

[0040] (d) an antibody to a polypeptide of the present invention,preferably to the polypeptide of SEQ ID NO:2.

[0041] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a disease, particularly 1)kidney-related diseases including diabetes, polycystic kidney disease,renal failure, etc. 2) brain-related diseases including Alzheimer'sdisease, stroke multiple sclerosis, neurodegenerative diseases, etc. 3)cardiovascular diseases including restenosis, myocardial infarction,dilated cardiomyopathy, atherosclerosis, etc. 4) others such asinflammation and cancer, amongst others.

[0042] The nucleotide sequences of the present invention are alsovaluable for chromosome identification. The sequence is specificallytargeted to, and can hybridize with, a particular location on anindividual human chromosome. The mapping of relevant sequences tochromosomes according to the present invention is an important firststep in correlating those sequences with gene associated disease. Once asequence has been mapped to a precise chromosomal location, the physicalposition of the sequence on the chromosome can be correlated withgenetic map data. Such data are found in, for example, V. McKusick,Mendelian Inheritance in Man (available on-line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

[0043] The differences in the cDNA or genomic sequence between affectedand unaffected individuals can also be determined. If a mutation isobserved in some or all of the affected individuals but not in anynormal individuals, then the mutation is likely to be the causativeagent of the disease.

[0044] The gene of the present invention inaps to human chromosome22q13.1.

[0045] The polypeptides of the invention or their fragments or analogsthereof, or cells expressing them, can also be used as immunogens toproduce antibodies immunospecific for polypeptides of the presentinvention. The term “immunospecific” means that the antibodies havesubstantially greater affinity for the polypeptides of the inventionthan their affinity for other related polypeptides in the prior art.

[0046] Antibodies generated against polypeptides of the presentinvention may be obtained by administering the polypeptides orepitope-bearing fragments, analogs or cells to an animal, preferably anon-human animal, using routine protocols. For preparation of monoclonalantibodies, any technique which provides antibodies produced bycontinuous cell line cultures can be used. Examples include thehybridoma technique (Kohler, G. and Milstein, C., Nature (1975)256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., Immunology Today (1983) 4:72) and the EBV-hybridomatechnique (Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp.77-96, Alan R. Liss, Inc., 1985).

[0047] Techniques for the production of single chain antibodies, such asthose described in U.S. Pat. No. 4,946,778, can also be adapted toproduce single chain antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms, including other mammals, may beused to express humanized antibodies.

[0048] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptide or to purify the polypeptidesby affinity chromatography.

[0049] Antibodies against polypeptides of the present invention may alsobe employed to treat the Diseases, amongst others.

[0050] In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

[0051] Another aspect of the invention relates to a method for inducingan immunological response in a mammal which comprises inoculating themammal with a polypeptide of the present invention, adequate to produceantibody and/or T cell immune response to protect said animal from theDiseases hereinbefore mentioned, amongst others. Yet another aspect ofthe invention relates to a method of inducing immunological response ina mammal which comprises, delivering a polypeptide of the presentinvention via a vector directing expression of the polynucleotide andcoding for the polypeptide in vivo in order to induce such animmunological response to produce antibody to protect said animal fromdiseases.

[0052] A further aspect of the invention relates to animmunological/ivaccine formulation (composition) which, when introducedinto a mammalian host, induces an immunological response in that mammalto a polypeptide of the present invention wherein the compositioncomprises a polypeptide or polynucleotide of the present invention. Thevaccine formulation may further comprise a suitable carrier. Since apolypeptide may be broken down in the stomach, it is preferablyadministered parenterally (for instance, subcutaneous, intramuscular,intravenous, or intradermal injection). Formulations suitable forparenteral administration include aqueous and non-aqueous sterileinjection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation instonic with theblood of the recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents or thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample, sealed ampoules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use. The vaccine formulation may also includeadjuvant systems for enhancing the immunogenicity of the formulation,such as oil-in water systems and other systems known in the art. Thedosage will depend on the specific activity of the vaccine and can bereadily determined by routine experimentation.

[0053] Polypeptides of the present invention are responsible for manybiological finctions, including many disease states, in particular theDiseases hereinbefore mentioned. It is therefore desirous to devisescreening methods to identify compounds which stimulate or which inhibitthe function of the polypeptide. Accordingly, in a further aspect, thepresent invention provides for a method of screening compounds toidentify those which stimulate or which inhibit the function of thepolypeptide. In general, agonists or antagonists may be employed fortherapeutic and prophylactic purposes for such Diseases as hereinbeforementioned. Compounds may be identified from a variety of sources, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. Such agonists, antagonists or inhibitors so-identifiedmay be natural or modified substrates, ligands, receptors, enzymes,etc., as the case may be, of the polypeptide; or may be structural orfunctional mimetics thereof (see Coligan et al., Current Protocols inImmunology 1(2):Chapter 5 (1991)).

[0054] The screening method may simply measure the binding of acandidate compound to the polypeptide, or to cells or membranes bearingthe polypeptide, or a fusion protein thereof by means of a labeldirectly or indirectly associated with the candidate compound.Alternatively, the screening method may involve competition with alabeled competitor. Further, these screening methods may test whetherthe candidate compound results in a signal generated by activation orinhibition of the polypeptide, using detection systems appropriate tothe cells bearing the polypeptide. Inhibitors of activation aregenerally assayed in the presence of a known agonist and the effect onactivation by the agonist by the presence of the candidate compound isobserved. Constitutively active polypeptides may be employed inscreening methods for inverse agonists or inhibitors, in the absence ofan agonist or inhibitor, by testing whether the candidate compoundresults in inhibition of activation of the polypeptide. Further, thescreening methods may simply comprise the steps of mixing a candidatecompound with a solution containing a polypeptide of the presentinvention, to form a mixture, measuring MT-MMP5-L activity in themixture, and comparing the MT-MMP5-L activity of the mixture to astandard. Fusion proteins, such as those made from Fc portion andMT-MMP5-L polypeptide, as hereinbefore described, can also be used forhigh-throughput screening assays to identify antagonists for thepolypeptide of the present invention (see D. Bennett et al., J MolRecognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995)).

[0055] The polynucleotides, polypeptides and antibodies to thepolypeptide of the present invention may also be used to configurescreening methods for detecting the effect of added compounds on theproduction of mRNA and polypeptide in cells. For example, an ELISA assaymay be constructed for measuring secreted or cell associated levels ofpolypeptide using monoclonal and polyclonal antibodies by standardmethods known in the art. This can be used to discover agents which mayinhibit or enhance the production of polypeptide (also called antagonistor agonist, respectively) from suitably manipulated cells or tissues.

[0056] The polypeptide may be used to identify membrane bound or solublereceptors, if any, through standard receptor binding techniques known inthe art. These include, but are not limited to, ligand binding andcrosslinking assays in which the polypeptide is labeled with aradioactive isotope (for instance, 1251), chemically modified (forinstance, biotinylated), or fused to a peptide sequence suitable fordetection or purification, and incubated with a source of the putativereceptor (cells, cell membranes, cell supernatants, tissue extracts,bodily fluids). Other methods include biophysical techniques such assurface plasmon resonance and spectroscopy. These screening methods mayalso be used to identify agonists and antagonists of the polypeptidewhich compete with the binding of the polypeptide to its receptors, ifany. Standard methods for conducting such assays are well understood inthe art.

[0057] Examples of potential polypeptide antagonists include antibodiesor, in some cases, oligonucleotides or proteins which are closelyrelated to the ligands, substrates, receptors, enzymes, etc., as thecase may be, of the polypeptide, e.g., a fragment of the ligands,substrates, receptors, enzymes, etc.; or small molecules which bind tothe polypeptide of the present invention but do not elicit a response,so that the activity of the polypeptide is prevented.

[0058] Thus, in another aspect, the present invention relates to ascreening kit for identifying agonists, antagonists, ligands, receptors,substrates, enzymes, etc. for polypeptides of the present invention; orcompounds which decrease or enhance the production of such polypeptides,which comprises:

[0059] (a) a polypeptide of the present invention;

[0060] (b) a recombinant cell expressing a polypeptide of the presentinvention;

[0061] (c) a cell membrane expressing a polypeptide of the presentinvention; or

[0062] (d) antibody to a polypeptide of the present invention;

[0063] which polypeptide is preferably that of SEQ ID NO:2.

[0064] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component.

[0065] It will be readily appreciated by the skilled artisan that apolypeptide of the present invention may also be used in a method forthe structure-based design of an agonist, antagonist or inhibitor of thepolypeptide, by:

[0066] (a) determining in the first instance the three-dimensionalstructure of the polypeptide;

[0067] (b) deducing the three-dimensional structure for the likelyreactive or binding site(s) of an agonist, antagonist or inhibitor;

[0068] (c) synthesing candidate compounds that are predicted to bind toor react with the deduced binding or reactive site; and

[0069] (d) testing whether the candidate compounds are indeed agonists,antagonists or inhibitors.

[0070] It will be further appreciated that this will normally be aninteractive process.

[0071] In a further aspect, the present invention provides methods oftreating abnormal conditions such as, for instance, 1) kidney-relateddiseases including diabetes, polycystic kidney disease, renal failure,etc. 2) brain-related diseases including Alzheimer's disease, strokemultiple sclerosis, neurodegenerative diseases, etc. 3) cardiovasculardiseases including restenosis, myocardial infarction, dilatedcardiomyopathy, atherosclerosis, etc. 4) others such as inflammation andcancer related to either an excess of, or an under-expression of,MT-MMP5-L polypeptide activity.

[0072] If the activity of the polypeptide is in excess, severalapproaches are available. One approach comprises administering to asubject in need thereof an inhibitor compound (antagonist) ashereinabove described, optionally in combination with a pharmaceuticallyacceptable carrier, in an amount effective to inhibit the function ofthe polypeptide, such as, for example, by blocking the binding ofligands, substrates, receptors, enzymes, etc., or by inhibiting a secondsignal, and thereby alleviating the abnormal condition. In anotherapproach, soluble forms of the polypeptides still capable of binding theligand, substrate, enzymes, receptors, etc. in competition withendogenous polypeptide may be administered. Typical examples of suchcompetitors include fragments of the MT-MMP5-L polypeptide.

[0073] In still another approach, expression of the gene encodingendogenous MT-MMP5-L polypeptide can be inhibited using expressionblocking techniques. Known such techniques involve the use of antisensesequences, either internally generated or separately administered (see,for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). Alternatively, oligonucleotides whichform triple helices with the gene can be supplied (see, for example, Leeet al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al., Science (1991) 251:1360). These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.

[0074] For treating abnormal conditions related to an under-expressionof MT-MMP5-L and its activity, several approaches are also available.One approach comprises administering to a subject a therapeuticallyeffective amount of a compound which activates a polypeptide of thepresent invention, i.e., an agonist as described above, in combinationwith a pharmaceutically acceptable carrier, to thereby alleviate theabnormal condition. Alternatively, gene therapy may be employed toeffect the endogenous production of MT-MMP5-L by the relevant cells inthe subject. For example, a polynucleotide of the invention may beengineered for expression in a replication defective retroviral vector,as discussed above. The retroviral expression construct may then beisolated and introduced into a packaging cell transduced with aretroviral plasmid vector containing RNA encoding a polypeptide of thepresent invention such that the packaging cell now produces infectiousviral particles containing the gene of interest. These producer cellsmay be administered to a subject for engineering cells in vivo andexpression of the polypeptide in vivo. For an overview of gene therapy,see Chapter 20, Gene Therapy and other Molecular Genetic-basedTherapeutic Approaches, (and references cited therein) in HumanMolecular Genetics, T Strachan and A P Read, BIOS Scientific PublishersLtd (1996). Another approach is to administer a therapeutic amount of apolypeptide of the present invention in combination with a suitablepharmaceutical carrier.

[0075] In a further aspect, the present invention provides forpharmaceutical compositions comprising a therapeutically effectiveamount of a polypeptide, such as the soluble form of a polypeptide ofthe present invention, agonist/antagonist peptide or small moleculecompound, in combination with a pharmaceutically acceptable carrier orexcipient. Such carriers include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

[0076] The composition will be adapted to the route of administration,for instance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, and the like.

[0077] The dosage range required depends on the choice of peptide orother compounds of the present invention, the route of administration,the nature of the formulation, the nature of the subject's condition,and the judgmnent of the attending practitioner. Suitable dosages,however, are in the range of 0.1-100 μg/kg of subject. Wide variationsin the needed dosage, however, are to be expected in view of the varietyof compounds available and the differing efficiencies of various routesof administration. For example, oral administration would be expected torequire higher dosages than administration by intravenous injection.Variations in these dosage levels can be adjusted using standardempirical routines for optimization, as is well understood in the art.

[0078] Polypeptides used in treatment can also be generated endogenouslyin the subject, in treatment modalities often referred to as “genetherapy” as described above. Thus, for example, cells from a subject maybe engineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

[0079] Polynucleotide and polypeptide sequences form a valuableinformation resource with which to identify further sequences of similarhomology. This is most easily facilitated by storing the sequence in acomputer readable medium and then using the stored data to search asequence database using well known searching tools, such as GCC.Accordingly, in a further aspect, the present invention provides for acomputer readable medium having stored thereon a polynucleotidecomprising the sequence of SEQ ID NO:1 and/or a polypeptide sequenceencoded thereby.

[0080] The following definitions are provided to facilitateunderstanding of certain terms used frequently hereinbefore.

[0081] “Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

[0082] “Isolated” means altered “by the hand of man” from the naturalstate. If an “isolated” composition or substance occurs in nature, ithas been changed or removed from its original environment, or both. Forexample, a polynucleotide or a polypeptide naturally present in a livinganimal is not “isolated,” but the same polynucleotide or polypeptideseparated from the coexisting materials of its natural state is“isolated” , as the term is employed herein.

[0083] “Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single-and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term “polynucleotide” also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

[0084] “Polypeptide” refers to any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds or modifiedpeptide bonds, i.e., peptide isosteres. “Polypeptide” refers to bothshort chains, commonly referred to as peptides, oligopeptides oroligomers, and to longer chains, generally referred to as proteins.Polypeptides may contain amino acids other than the 20 gene-encodedamino acids. “Polypeptides” include amino acid sequences modified eitherby natural processes, such as post-translational processing, or bychemical modification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications may occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentto the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from post-translation natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination (see, for instance, PROTEINS-STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993; Wold, F., Post-translational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., “Analysis for protein modifications and nonproteincofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et al., “ProteinSynthesis: Post-translational Modifications and Aging”, Ann NY Acad Sci(1992) 663:48-62).

[0085] “Variant” refers to a polynucleotide or polypeptide that differsfrom a reference polynucleotide or polypeptide, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

[0086] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity.

[0087] Preferred parameters for polypeptide sequence comparison includethe following:

[0088] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci.

[0089] USA. 89:10915-10919 (1992)

[0090] Gap Penalty: 12

[0091] Gap Length Penalty: 4

[0092] A program useful with these parameters is publicly available asthe “gap” program from Genetics Computer Group, Madison WI. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

[0093] Preferred parameters for polynucleotide comparison include thefollowing:

[0094] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970)

[0095] Comparison matrix: matches=+10, mismatch=0

[0096] Gap Penalty: 50

[0097] Gap Length Penalty: 3

[0098] Available as: The “gap” program from Genetics Computer Group,Madison Wis. These are the default parameters for nucleic acidcomparisons.

[0099] By way of example, a polynucleotide sequence of the presentinvention may be identical to the reference sequence of SEQ ID NO:1,that is be 100% identical, or it may include up to a certain integernumber of nucleotide alterations as compared to the reference sequence.Such alterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofnucleotide alterations is determined by multiplying the total number ofnucleotides in SEQ ID NO:1 by the numerical percent of the respectivepercent identity(divided by 100) and subtracting that product from saidtotal number of nucleotides in SEQ ID NO:1, or:

n _(n) ≦x _(n)−(x _(n) ·y),

[0100] wherein n_(n) is the number of nucleotide alterations, x_(n) isthe total number of nucleotides in SEQ ID NO:1, and y is, for instance,0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%,etc., and wherein any non-integer product of x_(n) and y is roundeddown to the nearest integer prior to subtracting it from x_(n).Alterations of a polynucleotide sequence encoding the polypeptide of SEQID NO:2 may create nonsense, missense or frameshift mutations in thiscoding sequence and thereby alter the polypeptide encoded by thepolynucleotide following such alterations.

[0101] Similarly, a polypeptide sequence of the present invention may beidentical to the reference sequence of SEQ ID NO:2, that is be 100%identical, or it may include up to a certain integer number of aminoacid alterations as compared to the reference sequence such that the %identity is less than 100%. Such alterations are selected from the groupconsisting of at least one amino acid deletion, substitution, includingconservative and non-conservative substitution, or insertion, andwherein said alterations may occur at the amino- or carboxy-terminalpositions of the reference polypeptide sequence or anywhere betweenthose terminal positions, interspersed either individually among theamino acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of amino acidalterations for a given % identity is determined by multiplying thetotal number of amino acids in SEQ ID NO:2 by the numerical percent ofthe respective percent identity(divided by 100) and then subtractingthat product from said total number of amino acids in SEQ ID NO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

[0102] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO:2, and y is, for instance0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein anynon-integer product of x_(a) and y is rounded down to the nearestinteger prior to subtracting it from x_(a).

[0103] “Fusion protein” refers to a protein encoded by two, oftenunrelated, fused genes or fragments thereof. In one example, EP-A-0 464discloses fusion proteins comprising various portions of constant regionof immunoglobulin molecules together with another human protein or partthereof. In many cases, employing an immunoglobulin Fc region as a partof a fusion protein is advantageous for use in therapy and diagnosisresulting in, for example, improved pharmacokinetic properties [see,e.g., EP-A 0232 262]. On the other hand, for some uses it would bedesirable to be able to delete the Fc part after the fusion protein hasbeen expressed, detected and purified.

[0104] All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

EXAMPLES

[0105] 1) Cloning of Full length Human MT-MMP5-L

[0106] Based on the partial EST sequence (HGS clone HCE3P83), afull-length cDNA clone was isolated by screening a human brain libraryusing PCR method. In order to confirm the start codon. a Lamda clonecontaining the genomic sequence of human MT-MMP5-L, was isolated from ahuman placenta genomic library. Therefore, the 5′ UTR sequence, whichcontains several stop codons, was revealed from the genomic clone, andconfirmed by RT-PCR of human brain mRNA. By comparison with the mouseMT5-MMP (D. Pei, JBC 274:8925-8932. 1999), the human MT-MMP5-L. we claimhere has a 82 amino acid deletion which encodes a signal peptide and apropeptide domain at the N-terminus. but it contains a completecatalytic domain. Thus, we define the sequence of the present inventionas a splicing variant of human MT-MMP5.

[0107] 2) Biological Studies of human MT-MMP5-L

[0108] Western Blot analysis was conducted in protein extracts ofvarious human tissues (Clontech Human Protein Medleys) using polyclonalantibodies generated against peptide fragments unique for MT-MMP-5 andnot present in MT-MMP-1, -2, -3, or -4. Protein expression was detectedin brain, kidney and heart. Western analysis was also conducted toevaluate MT-MMP-5 protein expression in normal and diabetic human kidneytissues. Results demonstrate significant up-regulation of MT-MMP-5protein expression in the diabetic kidney samples (n=8, each condition).!SEQUENCE INFORMATION SEQ ID NO:1CCGTTTCTAGCAGGAGGGGACACCAATAAAAGCTGCTCTC 40TGGAGATGAATTTGGCAGTGTCTGCAGGATGGGGTGAAGG 80AAAAAGAACTCAGAGCATGAGTCCAGGAGTCACTGGGGTC 120AAATGATCCCTCGTGTTCAAGGCACATCTCTGCCCAGGTA 160GAACAGAGCCTCTCCCAGCATAACGCATCTTTTTCTTTCC 200CGTGTCTTTCAGAACTGGTTAAAGTCCTATGGCTATCTGC 240TTCCCTATGACTCACGGGCATCTGCGCTGCACTCAGCGAA 280GGCCTTGCAGTCGGCAGTCTCCACTATGCAGCAGTTTTAC 320GGGATCCCGGTCACCGGTGTGTTGGATCAGACAACGATCG 360AGTGGATGAAGAAACCCCGATGTGGTGTCCCTGATCACCC 400CCACTTAAGCCGTAGGCGGAGAAACAAGCGCTATGCCCTG 440ACTGGACAGAAGTGGAGGCAAAAACACATCACCTACAGCA 480TTCACAACTATACCCCAAAAGTGGGTGAGCTAGACACGCG 520GAAAGCTATTCGCCAGGCTTTCGATGTGTGGCAGAAGGTG 560ACCCCACTGACCTTTGAAGAGGTGCCATACCATGAGATCA 600AAAGTGACCGGAAGGAGGCAGACATCATGATCTTTTTTGC 640TTCTGGTTTCCATGGCGACAGCTCCCCATTTGATGGAGAA 680GGGGGATTCCTGGCCCATGCCTACTTCCCTGGCCCAGGGA 720TTGGAGGAGACACCCACTTTGACTCCGATGAGCCATGGAC 760GCTAGGAAATGCCAACCATGACGGGAACGACCTCTTCCTG 800GTGGCTGTGCATGAGCTGGGCCACGCGCTGGGACTGGAGC 840ACTCCAGCGACCCCAGCGCCATCATGGCGCCCTTCTACCA 880GTACATGGAGACGCACAACTTCAAGCTGCCCCAGGACGAT 920CTCCAGGGCATCCAGAAGATCTATGGACCCCCAGCCGAGC 960CTCTGGAGCCCACAAGGCCACTCCCTACACTCCCCGTCCG 1000CAGGATCCACTCACCATCGGAGAGGAAACACGAGCGCCAG 1040CCCAGGCCCCCTCGGCCGCCCCTCGGGGACCGGCCATCCA 1080CACCAGGCACCAAACCCAACATCTGTGACGGCAACTTCAA 1120CACAGTGGCCCTCTTCCGGGGCGAGATGTTTGTCTTTAAG 1160GATCGCTGGTTCTGGCGTCTGCGCAATAACCGAGTGCAGG 1200AGGGCTACCCTATGCAGATCGAGCAGTTCTGGAAGGGCCT 1240GCCTGCCCGCATCGACGCAGCCTATGAAAGGGCCGATGGG 1280AGATTTGTCTTCTTCAAAGGTGACAAGTATTGGGTGTTTA 1320AGGAGGTGACgGTGGAGCCTGGGTACCCCCACAGCCTGGG 1360GGAGCTGGGCAGCTGTTTGCCCCGTGAAGGCATTGACACA 1400GCTCTGCGCTGGGAACCTGTGGGCAAGACCTACTTTTTCA 1440AAGGCGAGCGGTACTGGCGCTACAGCGAGGAGCGGCGGGC 1480CACGGACCCTGGCTACCCTAAGCCCATCACCGTGTGGAAG 1520GGCATCCCACAGGCTCCCCAAGGAGCCTTCATCAGCAAGG 1560AAGGATATTACACCTATTTCTACAAGGGCCGGGACTACTG 1600GAAGTCTGACAACCAGAAACTGAGCGTGGAGCCAGGCTAC 1640CCGCGCAACATCCTGCGTGACTGGATGGGCTGCAACCAGA 1680AGGAGGTGGAGCGGCGGAAGGAGCGGCGGCTGCCCCAGGA 1720CGACGTGGACATCATGGTGACCATCAACGATGTGCCGGGC 1760TCCGCGAACGCCGTGGCCGTGGTCATCCCCTGCATCCTGT 1800CCCTCTGCATCCTGGTGCTGGTCTACACCATCTTCCAGTT 1840CAAGAACAAGACAGGCCCTCAGCCTGTCACCTACTATAAG 1880CGGCCAGTCCAGGAATGGGTGTGAGCAGCCCAGAGCCCTC 1920 TCT 1923 SEQ ID NO:2MQQFYGIPVTGVLDQTTIEWMKKPRCGVPDHPHLSRRRRN 40KRYALTGQKWRQKHITYSIHNYTPKVGELDTRKAIRQAFD 80VWQKVTPLTFEEVPYHEIKSDRKEADIMIFFASGFHGDSS 120PFDGEGGFLAHAYFPGPGIGGDTHFDSDEPWTLGNANHDG 160NDLFLVAVHELGHALGLEHSSDPSAIMAPFYQYMETHNFK 200LPQDDLQGIQKIYGPPAEPLEPTRPLPTLPVRRIHSPSER 240KHERQPRPPRPPLGDRPSTPGTKPNICDGNFNTVALFRGE 280MFVFKDRWFWRLRNNRVQEGYPMQIEQFWKGLPARIDAAY 320ERADGRFVFFKGDKYWVFKEVTVEPGYPHSLGELGSCLPR 360EGIDTALRWEPVGKTYFFKGERYWRYSEERRATDPGYPKP 400ITVWKGIPQAPQGAFISKEGYYTYFYKGRDYWKSDNQKLS 440VEPGYPRNILRDWMGCNQKEVERRKERRLPQDDVDIMVTI 480NDVPGSANAVAVVIPCILSLCILVLVYTIFQFKNKTGPQP 520 VTYYKRPVQEWV 532

[0109]

1 2 1 1923 DNA HOMO SAPIENS 1 ccgtttctag caggagggga caccaataaaagctgctctc tggagatgaa tttggcagtg 60 tctgcaggat ggggtgaagg aaaaagaactcagagcatga gtccaggagt cactggggtc 120 aaatgatccc tcgtgttcaa ggcacatctctgcccaggta gaacagagcc tctcccagca 180 taacgcatct ttttctttcc cgtgtctttcagaactggtt aaagtcctat ggctatctgc 240 ttccctatga ctcacgggca tctgcgctgcactcagcgaa ggccttgcag tcggcagtct 300 ccactatgca gcagttttac gggatcccggtcaccggtgt gttggatcag acaacgatcg 360 agtggatgaa gaaaccccga tgtggtgtccctgatcaccc ccacttaagc cgtaggcgga 420 gaaacaagcg ctatgccctg actggacagaagtggaggca aaaacacatc acctacagca 480 ttcacaacta taccccaaaa gtgggtgagctagacacgcg gaaagctatt cgccaggctt 540 tcgatgtgtg gcagaaggtg accccactgacctttgaaga ggtgccatac catgagatca 600 aaagtgaccg gaaggaggca gacatcatgatcttttttgc ttctggtttc catggcgaca 660 gctccccatt tgatggagaa gggggattcctggcccatgc ctacttccct ggcccaggga 720 ttggaggaga cacccacttt gactccgatgagccatggac gctaggaaat gccaaccatg 780 acgggaacga cctcttcctg gtggctgtgcatgagctggg ccacgcgctg ggactggagc 840 actccagcga ccccagcgcc atcatggcgcccttctacca gtacatggag acgcacaact 900 tcaagctgcc ccaggacgat ctccagggcatccagaagat ctatggaccc ccagccgagc 960 ctctggagcc cacaaggcca ctccctacactccccgtccg caggatccac tcaccatcgg 1020 agaggaaaca cgagcgccag cccaggccccctcggccgcc cctcggggac cggccatcca 1080 caccaggcac caaacccaac atctgtgacggcaacttcaa cacagtggcc ctcttccggg 1140 gcgagatgtt tgtctttaag gatcgctggttctggcgtct gcgcaataac cgagtgcagg 1200 agggctaccc tatgcagatc gagcagttctggaagggcct gcctgcccgc atcgacgcag 1260 cctatgaaag ggccgatggg agatttgtcttcttcaaagg tgacaagtat tgggtgttta 1320 aggaggtgac ggtggagcct gggtacccccacagcctggg ggagctgggc agctgtttgc 1380 cccgtgaagg cattgacaca gctctgcgctgggaacctgt gggcaagacc tactttttca 1440 aaggcgagcg gtactggcgc tacagcgaggagcggcgggc cacggaccct ggctacccta 1500 agcccatcac cgtgtggaag ggcatcccacaggctcccca aggagccttc atcagcaagg 1560 aaggatatta cacctatttc tacaagggccgggactactg gaagtctgac aaccagaaac 1620 tgagcgtgga gccaggctac ccgcgcaacatcctgcgtga ctggatgggc tgcaaccaga 1680 aggaggtgga gcggcggaag gagcggcggctgccccagga cgacgtggac atcatggtga 1740 ccatcaacga tgtgccgggc tccgcgaacgccgtggccgt ggtcatcccc tgcatcctgt 1800 ccctctgcat cctggtgctg gtctacaccatcttccagtt caagaacaag acaggccctc 1860 agcctgtcac ctactataag cggccagtccaggaatgggt gtgagcagcc cagagccctc 1920 tct 1923 2 532 PRT HOMO SAPIENS 2Met Gln Gln Phe Tyr Gly Ile Pro Val Thr Gly Val Leu Asp Gln Thr 1 5 1015 Thr Ile Glu Trp Met Lys Lys Pro Arg Cys Gly Val Pro Asp His Pro 20 2530 His Leu Ser Arg Arg Arg Arg Asn Lys Arg Tyr Ala Leu Thr Gly Gln 35 4045 Lys Trp Arg Gln Lys His Ile Thr Tyr Ser Ile His Asn Tyr Thr Pro 50 5560 Lys Val Gly Glu Leu Asp Thr Arg Lys Ala Ile Arg Gln Ala Phe Asp 65 7075 80 Val Trp Gln Lys Val Thr Pro Leu Thr Phe Glu Glu Val Pro Tyr His 8590 95 Glu Ile Lys Ser Asp Arg Lys Glu Ala Asp Ile Met Ile Phe Phe Ala100 105 110 Ser Gly Phe His Gly Asp Ser Ser Pro Phe Asp Gly Glu Gly GlyPhe 115 120 125 Leu Ala His Ala Tyr Phe Pro Gly Pro Gly Ile Gly Gly AspThr His 130 135 140 Phe Asp Ser Asp Glu Pro Trp Thr Leu Gly Asn Ala AsnHis Asp Gly 145 150 155 160 Asn Asp Leu Phe Leu Val Ala Val His Glu LeuGly His Ala Leu Gly 165 170 175 Leu Glu His Ser Ser Asp Pro Ser Ala IleMet Ala Pro Phe Tyr Gln 180 185 190 Tyr Met Glu Thr His Asn Phe Lys LeuPro Gln Asp Asp Leu Gln Gly 195 200 205 Ile Gln Lys Ile Tyr Gly Pro ProAla Glu Pro Leu Glu Pro Thr Arg 210 215 220 Pro Leu Pro Thr Leu Pro ValArg Arg Ile His Ser Pro Ser Glu Arg 225 230 235 240 Lys His Glu Arg GlnPro Arg Pro Pro Arg Pro Pro Leu Gly Asp Arg 245 250 255 Pro Ser Thr ProGly Thr Lys Pro Asn Ile Cys Asp Gly Asn Phe Asn 260 265 270 Thr Val AlaLeu Phe Arg Gly Glu Met Phe Val Phe Lys Asp Arg Trp 275 280 285 Phe TrpArg Leu Arg Asn Asn Arg Val Gln Glu Gly Tyr Pro Met Gln 290 295 300 IleGlu Gln Phe Trp Lys Gly Leu Pro Ala Arg Ile Asp Ala Ala Tyr 305 310 315320 Glu Arg Ala Asp Gly Arg Phe Val Phe Phe Lys Gly Asp Lys Tyr Trp 325330 335 Val Phe Lys Glu Val Thr Val Glu Pro Gly Tyr Pro His Ser Leu Gly340 345 350 Glu Leu Gly Ser Cys Leu Pro Arg Glu Gly Ile Asp Thr Ala LeuArg 355 360 365 Trp Glu Pro Val Gly Lys Thr Tyr Phe Phe Lys Gly Glu ArgTyr Trp 370 375 380 Arg Tyr Ser Glu Glu Arg Arg Ala Thr Asp Pro Gly TyrPro Lys Pro 385 390 395 400 Ile Thr Val Trp Lys Gly Ile Pro Gln Ala ProGln Gly Ala Phe Ile 405 410 415 Ser Lys Glu Gly Tyr Tyr Thr Tyr Phe TyrLys Gly Arg Asp Tyr Trp 420 425 430 Lys Ser Asp Asn Gln Lys Leu Ser ValGlu Pro Gly Tyr Pro Arg Asn 435 440 445 Ile Leu Arg Asp Trp Met Gly CysAsn Gln Lys Glu Val Glu Arg Arg 450 455 460 Lys Glu Arg Arg Leu Pro GlnAsp Asp Val Asp Ile Met Val Thr Ile 465 470 475 480 Asn Asp Val Pro GlySer Ala Asn Ala Val Ala Val Val Ile Pro Cys 485 490 495 Ile Leu Ser LeuCys Ile Leu Val Leu Val Tyr Thr Ile Phe Gln Phe 500 505 510 Lys Asn LysThr Gly Pro Gln Pro Val Thr Tyr Tyr Lys Arg Pro Val 515 520 525 Gln GluTrp Val 530

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: (i) an isolated polypeptide comprising an amino acidsequence selected from the group having at least: (a) 70% identity; (b)80% identity; (c) 90% identity; or (d) 95% identity; to the amino acidsequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2; (ii) anisolated polypeptide comprising the amino acid sequence of SEQ ID NO:2;or (iii) an isolated polypeptide which is the amino acid sequence of SEQID NO:2.
 2. An isolated polynucleotide selected from the groupconsisting of: (i) an isolated polynucleotide comprising a nucleotidesequence encoding a polypeptide that has at least (a) 70% identity; (b)80% identity; (c) 90% identity; or (d) 95% identity; to the amino acidsequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2; (ii) anisolated polynucleotide comprising a nucleotide sequence that has atleast: (a) 70% identity (b) 80% identity; (c) 90% identity; or (d) 95%identity; over its entire length to a nucleotide sequence encoding thepolypeptide of SEQ ID NO:2; (iii) an isolated polynucleotide comprisinga nucleotide sequence which has at least: (a) 70% identity; (b) 80%identity; (c) 90% identity; or (d) 95% identity; to that of SEQ ID NO: 1over the entire length of SEQ ID NO: 1; (iv) an isolated polynucleotidecomprising a nucleotide sequence encoding the polypeptide of SEQ ID NO:2; (v) an isolated polynucleotide which is the polynucleotide of SEQ IDNO: 1; or (vi) an isolated polynucleotide obtainable by screening anappropriate library under stringent hybridization conditions with alabeled probe having the sequence of SEQ ID NO: 1 or a fragmentthereof.; or a nucleotide sequence complementary to said isolatedpolynucleotide.
 3. An antibody immunospecific for the polypeptide ofclaim
 1. 4. A method for the treatment of a subject: (i) in need ofenhanced activity or expression of the polypeptide of claim 1comprising: (a) administering to the subject a therapeutically effectiveamount of an agonist to said polypeptide; and/or (b) providing to thesubject an isolated polynucleotide comprising a nucleotide sequenceencoding said polypeptide in a form so as to effect production of saidpolypeptide activity in vivo.; or (ii) having need to inhibit activityor expression of the polypeptide of claim 1 comprising: (a)administering to the subject a therapeutically effective amount of anantagonist to said polypeptide; and/or (b) administering to the subjecta nucleic acid molecule that inhibits the expression of a nucleotidesequence encoding said polypeptide; and/or (c) administering to thesubject a therapeutically effective amount of a polypeptide thatcompetes with said polypeptide for its ligand, substrate , or receptor.5. A process for diagnosing a disease or a susceptibility to a diseasein a subject related to expression or activity of the polypeptide ofclaim 1 in a subject comprising: (a) determining the presence or absenceof a mutation in the nucleotide sequence encoding said polypeptide inthe genome of said subject; and/or (b) analyzing for the presence oramount of said polypeptide expression in a sample derived from saidsubject.
 6. A method for screening to identify compounds which stimulateor which inhibit the function of the polypeptide of claim 1 whichcomprises a method selected from the group consisting of: (a) measuringthe binding of a candidate compound to the polypeptide (or to the cellsor membranes bearing the polypeptide) or a fusion protein thereof bymeans of a label directly or indirectly associated with the candidatecompound; (b) measuring the binding of a candidate compound to thepolypeptide (or to the cells or membranes bearing the polypeptide) or afusion protein thereof in the presence of a labeled competitor; (c)testing whether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide, using detection systemsappropriate to the cells or cell membranes bearing the polypeptide; (d)mixing a candidate compound with a solution containing a polypeptide ofclaim 1, to form a mixture, measuring activity of the polypeptide in themixture, and comparing the activity of the mixture to a standard; or (e)detecting the effect of a candidate compound on the production of mRNAencoding said polypeptide and said polypeptide in cells, using forinstance, an ELISA assay.
 7. An agonist or an antagonist of thepolypeptide of claim
 1. 8. An expression system comprising apolynucleotide capable of producing a polypeptide of claim 1 when saidexpression system is present in a compatible host cell.
 9. A process forproducing a recombinant host cell comprising transforming ortransfecting a cell with the expression system of claim 8 such that thehost cell, under appropriate culture conditions, produces a polypeptidecomprising an amino acid sequence having at least 70% identity to theamino acid sequence of SEQ ID NO:2 over the entire length of SEQ IDNO:2.
 10. A recombinant host cell produced by the process of claim 9.11. A membrane of a recombinant host cell of claim 10 expressing apolypeptide comprising an amino acid sequence having at least 70%identity to the amino acid sequence of SEQ ID NO:2 over the entirelength of SEQ ID NO:2.
 12. A process for producing a polypeptidecomprising culturing a host cell of claim 10 under conditions sufficientfor the production of said polypeptide and recovering the polypeptidefrom the culture.